1. Field of the Invention
The present invention relates to methods, kits, primers, probes, and systems for distinguishing between nucleotide variants that are close in proximity on a gene. More particularly, aspects of the present invention relate to methods, kits, primers, probes, and systems for using a small amplicon assay in combination with unlabeled probes in conducting a high resolution thermal melting analysis of a biological sample containing a locus of interest in order to discern between disease-causing and benign variants that are close in proximity on a gene. The present invention also relates to methods of detecting a disease in a patient based on the patient's genotype by determining whether the patient has a disease-causing variant at a locus of interest on the patient's genome.
2. Description of the Background
Melt curve analysis or high resolution thermal melting is an important technique for analyzing nucleic acids. In accordance with some methods, a double stranded nucleic acid is denatured in the presence of a dye that indicates whether the two strands are bound or not. Examples of such indicator dyes include non-specific binding dyes such as SYBR® Green I, whose fluorescence efficiency depends strongly on whether it is bound to double stranded DNA. As the temperature of the mixture is increased, a reduction in fluorescence from the dye indicates that the nucleic acid molecule has melted, i.e., unzipped, partially or completely. Thus, by measuring the dye fluorescence as a function of temperature, information is gained regarding the length of the duplex, the GC content or even the exact sequence. See, e.g., Ririe et al. (Anal Biochem 245:154-160, 1997), Wittwer et al. (Clin Chem 49:853-860, 2003), Liew et al. (Clin Chem 50:1156-1164 (2004), Herrmann et al. (Clin Chem 52:494-503, 2006), Knapp et al. (U.S. Patent Application Publication No. 2002/0197630), Wittwer et al. (U.S. Patent Application Publication No. 2005/0233335), Wittwer et al. (U.S. Patent Application Publication No. 2006/0019253), Sundberg et al. (U.S. Patent Application Publication No. 2007/0026421) and Knight et al. (U.S. Patent Application Publication No. 2007/0231799).
A number of commercial instruments exist that perform thermal melts on DNA. Examples of available instruments include the Idaho Technology HR-1 high resolution melter and the Idaho Technology LightScanner high resolution melter. The HR-1 high resolution melter has a high resolution fluorescent signal to noise ratio and temperature resolution. However, it suffers from a limitation that it can only analyze one sample at a time, and the sample container must be replaced manually. Replacement of the container for each test may contribute to run-to-run temperature variability. The LightScanner high resolution melter also has good signal and temperature resolution, and operates on a 96-well plate sample container. A typical mode of operation for these analyzers is to apply heat to the sample(s) in a controlled manner to achieve a linear rise in temperature versus time. Simultaneously, a stable continuous fluorescence excitation light is applied, and emitted fluorescence is collected continuously over fixed integration time intervals. The fluorescence intensity data is converted from a time basis to a temperature basis based on the knowledge of the temperature ramp versus time.
In addition to such commercial instruments, microfluidic systems have also been developed for performing thermal melt analysis. For example, Sundberg et al. (U.S. Patent Application Publication No. 2007/0026421) and Knight et al. (U.S. Patent Application Publication No. 2007/0231799), each incorporated by reference herein, describe methods, systems, kits and devices for conducting binding assays using molecular melt curves in microfluidic devices. Molecule(s) to be assayed can be flowed through microchannels in the devices where the molecule(s) optionally are exposed to additional molecules constituting, e.g., fluorescence indicator molecules and/or binding partners of the molecule being assayed. The molecules involved are then heated (and/or cooled) and a detectable property of the molecules is measured over a range of temperatures. From the resulting data, a thermal property curve(s) is constructed which allows determination and quantification of the binding affinity of the molecules involved. Other microfluidic systems useful for thermal melting analysis are described in Hasson et al. (U.S. Patent Application Publication No. 2009/0248349), Hasson et al. (U.S. Patent Application Publication No. 2009/0318306), Hasson et al. (U.S. Patent Application Publication No. 2009/0324037), Cao (U.S. Patent Application Publication No. 2010/0233687) and Coursey (U.S. Patent Application Publication No. 2011/0056926).
Although high resolution thermal melting is a useful tool for genotyping, many genotyping assay attempts do not present a significant difference in the melt curves of targeted variants, especially variants that are in close proximity. For example, prior techniques for Cystic Fibrosis (CF) testing using high resolution thermal melt analysis lacked the ability to discern between benign and disease-causing variants in close proximity in Exon 10 of the CFTR (cystic fibrosis transmembrane conductance regulator) gene.
Cystic fibrosis, also known as mucoviscidosis, is the most common lethal autosomal recessive disorder and the most common life-shortening inherited diseases among the Caucasian population. Cystic Fibrosis is caused by mutations of the CFTR gene. This disease affects multiple systems and organs in the body, including the lungs, pancreas, intestines, and liver, and occurs in 1 in 2,500 Caucasian newborns (Rowntree and Harris, Annals of Human Genetics. 2003; 67:471-485). Currently, more than 30,000 children and young adults are affected by Cystic Fibrosis in the United States. The ΔF508 mutation, a three base pair deletion that removes a phenylalanine residue at amino acid position 508 (ΔF508), is the mutation occurring on the majority of CF chromosomes being found on 70%-75% of North American CF chromosomes (Kerem et al., Science. 1989; 245:1073-1080). This three base pair deletion affects the cytoplasmic nucleotide-binding domain (NBD-1) and causes severe dysfunction of chloride transportation a cross cellular membranes.
Since ΔF508 was first identified in CF gene by Kerem and his colleagues using restriction fragment length polymorphisms (RFLP), more techniques have been used to explore CF mutations, such as a restriction map of the genomic clone (Chou et al., J. Biol. Chem. 1991; 266:24471-24476), denaturing gel gradient electrophoresis (DGGE) using PCR (Pallares-Ruiz et al. Human Reproduction. 1999; 14:3035-3040), allele-specific primers and fluorometry (Litia et al. Genome Res. 1992; 2:157-162), DNA sequencing (Kerem et al. Pediatrics. 1997; 100:1-6), and saturated dye and melting analysis (Zhou et al. Clin. Chem. 2008; 54:1648-1656). These techniques enhanced the understanding of the structures of the CFTR gene and CFTR mutations related to the CF disease, providing a promising outlook for early diagnosis and treatments to the CF patients.
However, it has been discovered that the benign variants F508C, I507V, and I506V that neighbor ΔF508/ΔI507, often present similar genotype patterns and are mistakenly recognized as the disease-causing variants ΔF508 or ΔI507 (Desgeorges et al. Am J Hum Genet. 1994; 54:384-5). The substitution of cysteine for phenylalanine 508 (F508C) and substitution of valine for isoleucine 506 (I506V) have been reported to be homologous for ΔF508 mutation and are difficult to be distinguished from ΔF508 (Kobayashi et al. Am. J. Hum. Genet. 1990; 47:611-615). ΔI507, a three base pair in frame deletion close to ΔF508 that results in the deletion of isoleucine, has been reported to present the same genotyping pattern as I506V, a benign variant at the same location of ΔI507 (Johnson et al., J Mol Des 2007; 9:401-407).
Although commercial kits are available to detect CF mutations, these kits do not make it possible to distinguish between all benign and disease-causing mutations in Exon 10 in a rapid detection system (Johnson et. al., J. Mol. Diagn. 2007. 9:401-407). It is critical and necessary for researchers, physicians, and diagnostic laboratories to be able to differentiate ΔF508 and ΔI507 from F508C, I507V, and I506V in order to reliably detect and diagnose disease-causing genotypes.
Accordingly, there is a need in the art for reliable methods, kits, probes, and systems that will be useful in discerning between nucleotide sequence variants that present similar genotype patterns. Similarly, there is a need for methods, kits, probes, and systems for accurately detecting a disease in a patient when a disease-causing variant may be mistakenly recognized as a benign variant because the variants present similar melting signatures.